Immunofluorescence is a method to detect the distribution of an antigen in a biological sample through the specific binding of an antibody which itself is coupled to a fluorescent agent. The antibody binds specifically to the target molecule so that the fluorescent label qualitatively and/or quantitatively reports the presence of the target.
Different methods for the attachment of the fluorescent label have been developed. The direct labeling method utilizes a primary antibody (an antibody that recognizes the target) which is then coupled to the fluorescent agent. This method is labor-intensive and a certain amount of antibody might be inactivated in the process (if the label attaches itself to the antigen recognizing region of the antibody).
The indirect method utilizes a secondary antibody—an antibody which recognizes the primary antibody—coupled to a fluorescent agent to attach the label. Several different variations of this method have been described. Often the primary antibody is applied first to the sample, followed by a washing step and the application of a species specific secondary antibody which carries a fluorescent label. This often results in background problems due to unspecific binding of the secondary antibody to the tissue. Another approach has utilized preformed primary-secondary antibody complexes (Tuson et al. 1990). This method enables indirect labeling of primary antibodies derived from the same species, but the use of divalent secondary antibodies can lead to crosslinked complexes.
A similar approach which avoids this problem is the use of monovalent Fc specific Fab fragments for the generation of pre-formed complexes and has been commercialized by Molecular Probes (Zenon; Eugene, Oreg.). Others have modified this method by utilizing Fab fragments that recognize both the Fc and F(ab′)2 regions of the primary antibody (Brown et al. 2004). This approach is described in U.S. application Ser. No. 10/118,204 filed Apr. 5, 2002 which is incorporated herein by reference.
All the methods above have in common that the primary antibody is labeled before it is contacted with a biological sample. The primary antibody is either directly labeled with a fluorophore (through chemical means, by binding to secondary antibody or a Fab fragment labeled with a fluorescent agent) or that the primary antibodies are detected with a bivalent secondary antibody which is labeled with a fluorophore (usually by chemical means). This can cause problems especially in fixed, paraffin embedded tissue specimen which is generally less accessible to large molecules than other biological samples (e.g. fixed, permeabilized cultured cells). For example, an antibody in pre-formed complex with a secondary antibody has twice the molecular weight of the primary antibody. An antibody in a pre-formed complex with Fab fragments has about twice the molecular weight of the primary antibody if three Fab fragments are bound per antibody.
The increased size of these complexes can prevent sufficient penetration of the tissue and thus restrict or inhibit detection of the target molecule, cause excessive background staining and thus complicate the detection of the target molecule. This is especially relevant for the detection of a nuclear biomarker, where the antibody—Fab complexes are unable to penetrate the complexed protein of the nucleus and instead are found accumulated in the cytoplasm. Thus, a need exists for improved methods of analyzing intracellular antigens.
There is a growing body of evidence that tumor cell proliferation has prognostic significance for a variety of commonly occurring malignancies, including lymphoma (Braylan R. C., Diamond L. W., Powell M. L., Harty-Golder B. Percentage of cells in the S phase of the cell cycle in human lymphoma determined by flow cytometry: Correlation with labeling index and patient survival. Cytometry 1980; 1:171-174; and Bauer K. D., Merkel D. E., Winter J. N., et al. Prognostic implications of ploidy and proliferative activity in diffuse large cell lymphomas. Cancer Res 1986; 46:3173-3178), breast cancer (Clark G. M., Dressler L. G., Owens M. A., Pounds G., Oldaker T., McGuire W. L. Prediction of relapse or survival in patients with node-negative breast cancer by DNA flow cytometry. N Engl J Med 1989; 320:627-633; Silvestrini R., Daidone M. G., Gasparini G. Cell kinetics as a prognostic marker in node-negative breast cancer. Cancer 1985; 56:1982-1987; and Sigurdsson H., Baldetorp B., Borg A., et al. Indicators of prognosis in node-negative breast cancer. N Engl J Med 1990; 322:1045-1053), and colon cancer (Bauer K. D., Lincoln S. T., Vera-Roman J. M., et al. Prognostic implications of proliferative activity and DNA aneuploidy in colonic adenocarcinomas. Lab Invest 1987; 57:329-335). In some studies, tumor cell proliferation has independent prognostic significance, even if total DNA content analysis (“ploidy”) does not (Visscher D. W., Zarbo R. J., Greenawald K. A., Crissman J. D. Prognostic significance of morphological parameters and flow cytometric DNA analysis in carcinoma of the breast. Pathol Ann 1990; 25(Part-I): 171-210).
Flow cytometry (FCM) has been used extensively to determine cell cycle activity, primarily by quantitation of the S-phase portion of the DNA content analysis (“ploidy”). This method suffers from a number of serious technical limitations, however. First, it may be difficult to obtain single cell suspensions from solid tumors, and variable numbers of tumor cells may be lost during preparation. Second, the tumor cells are variably diluted by benign normal and inflammatory cells, which can lead to underestimation of the S-phase fraction, particularly for DNA diploid tumors. Third, the complexity of the DNA content analysis (“ploidy”), which consists of a series of overlapping curves, may preclude the accurate use of curve-fitting algorithms to measure the S-phase portion of the histogram. Multicenter studies have shown poor reproducibility for flow-cytometric S-phase fraction, making the practical clinical usefulness of the measurement somewhat doubtful. Another problem associated with cell kinetic measurement by flow cytometry is that only the S-phase fraction is typically determined, whereas a significant proportion of the tumor cell population may reside in the G1 phase of the cell cycle, comprised of cells committed to entering the cycle but not yet synthesizing DNA. Conceivably, two tumors may have identical S-phase fractions but differ significantly in the total fraction of cells in the nonresting state, and thus may exhibit different growth kinetics and response to cycle-dependent chemotherapeutic agents.
For all of these reasons, in situ methods of tumor cell cycle analysis may provide more biologically meaningful information than can be obtained using disaggregated tumor cells (Weinberg I. S. Relative applicability of image analysis and flow cytometry in clinical medicine. In: Bauer K. D., Duque R. E., eds. Flow cytometry: Principles and applications. Baltimore: Williams and Wllkins; 1992:359-372; and Weinberg D. S. Proliferation indices in solid tumors. Adv Pathol Lab Med 1992; 5:163-191). In addition to guaranteeing that the acquired measurements are made specifically on the tumor cells, in situ methods can allow more widespread sampling of the tumor and determination of tumor cell heterogeneity.